The amount of BCR/TCR template RNA can vary between samples, even when identical input RNA amount is used. For this reason, the optimal PCR2 cyclecount (N° of cycles at which a sufficient amount of product is generated without exhausting the reagents) must be determined for each sample individually. The optimal PCR2 cyclecount is defined as the cycle at which 2/3rds of the maximal fluorescence is reached.
- Input data: Gene expression result and Cq result files (.csv format) from CFX96 Real-Time System.
- Parameters: Wells to filter from analysis; Samples to highlight; Cycle cutoff values; ...
- Outputs: PDF file with relevant metadata, amplification plots, and optimal cyclecount for each sample.
Once the libraries have been prepped, their size distribution must be assessed to ensure that they are free from non-specific amplicons. This step also allows to estimate the library concentrations.
- Input data: TapeStation .XML results file, Electropherogram .csv file, and annotations .csv file if non-standard sample loading order.
- Parameters: Wells to exclude, dilution factor, Reference sizes of BCR/TCR/IGH peaks.
- Outputs: PDF file with relevant metadata, size distribution and concentration estimations.
The last step of libraryprep is to accurately quantify the libraries. This is acchieved through qPCR with standards.
- Input data: Gene expression result and Cq result files (.csv format) from CFX96 Real-Time System. The standard names should correspond to the quantity, the well target should be 'ladder'.
- Parameters: Wells to exclude, dilution factor, Reference sizes of BCR/TCR/IGH peaks.
- Outputs: PDF file with relevant metadata, amplification curves, and absolute sample concentrations with SD.